WO2019117812A1 - Sondes moléculaires en proche infrarouge destinées à être utilisées dans le diagnostic de troubles fibrotiques et le criblage de médicaments anti-fibrotiques - Google Patents

Sondes moléculaires en proche infrarouge destinées à être utilisées dans le diagnostic de troubles fibrotiques et le criblage de médicaments anti-fibrotiques Download PDF

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WO2019117812A1
WO2019117812A1 PCT/SG2018/050608 SG2018050608W WO2019117812A1 WO 2019117812 A1 WO2019117812 A1 WO 2019117812A1 SG 2018050608 W SG2018050608 W SG 2018050608W WO 2019117812 A1 WO2019117812 A1 WO 2019117812A1
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compound
fnp1
fibrotic
cells
scarring
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PCT/SG2018/050608
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Kanyi Pu
Chenjie Xu
Qingqing MIAO
Chen Loong David YEO
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Nanyang Technological University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/444—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a six-membered ring with nitrogen as a ring heteroatom, e.g. amrinone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0028—Oxazine dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
    • A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
    • A61K49/0039—Coumarin dyes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0013—Luminescence
    • A61K49/0017—Fluorescence in vivo
    • A61K49/005—Fluorescence in vivo characterised by the carrier molecule carrying the fluorescent agent
    • A61K49/0052—Small organic molecules
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B11/00—Diaryl- or thriarylmethane dyes
    • C09B11/04—Diaryl- or thriarylmethane dyes derived from triarylmethanes, i.e. central C-atom is substituted by amino, cyano, alkyl
    • C09B11/10—Amino derivatives of triarylmethanes
    • C09B11/24—Phthaleins containing amino groups ; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B23/00—Methine or polymethine dyes, e.g. cyanine dyes
    • C09B23/10—The polymethine chain containing an even number of >CH- groups
    • C09B23/105—The polymethine chain containing an even number of >CH- groups two >CH- groups
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09B—ORGANIC DYES OR CLOSELY-RELATED COMPOUNDS FOR PRODUCING DYES, e.g. PIGMENTS; MORDANTS; LAKES
    • C09B57/00—Other synthetic dyes of known constitution
    • C09B57/02—Coumarine dyes

Definitions

  • the current invention relates to molecular probes that can be used in the fields of diagnosis and screening.
  • the current invention also relates to the discovery of anti-fibrotic activity in a number of known compounds, where such activity was not known.
  • Abnormal scarring is a result of aberrant wound healing and may develop following any insult to the deep dermis like burn injury, lacerations, abrasions, surgery, piercings and vaccinations.
  • a total of 100 million patients develop scars each year as a result of 55 million elective surgeries and 25 million operations after trauma. Therefore, the detection of skin diseases at an early stage is critical to their timely treatment. For example, as keloids, which are a fibrous scar lesions, overgrow their wound boundaries due to over-exuberant healing following skin injury they can cause limited joint mobility, psychological distress and significant pain and itch to those afflicted.
  • Drug discovery and development involves the screening of compounds for their specificity and efficacy. There is currently no dedicated screening method to identify drugs for abnormal scarring. Drug screening from a library of compounds can be performed through mass spectrometry, genomic/genetic analysis, cell reporter genes and even computational modeling. However, these methods are limited by the requirement for specialist training (i.e. computational modeling) as well as laborious processes. Thus, there also remains a need for such a dedicated screening test.
  • X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom or an NH group;
  • Y is a self-immolative linking group
  • Z represents a peptide group of formula la:
  • the wavy line represents the point of attachment to the rest of the molecule
  • R 1 represents Ci-e alkyl or OR 2 ;
  • R 2 represents Ci-e alkyl that is unsubstituted or substituted by one or more groups selected from halo, C1 -3 alkyl and aryl;
  • n 1 or 2;
  • n 0 or 1 , or pharmaceutically acceptable salts or solvates thereof, provided that:
  • X may be independently selected from the group consisting of:
  • X may be independently selected from the group consisting of:
  • Z may be independently selected from the group consisting of:
  • Y when present, Y may be independently selected from the group consisting of:
  • Y may be independently selected from the 5 group consisting of:
  • the compound formula I may be selected from:
  • the compound of formula I may be selected from:
  • a method of detecting a fibrotic condition in a subject comprising the steps of providing a compound of formula I, as defined in the first aspect and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof to a subject, subjecting a tissue or organ suspected of suffering from a fibrotic condition to irradiation with light and detecting fluorescence from the irradiated tissue or organ, wherein an increase in fluorescence compared to a control indicates the presence of a fibrotic condition.
  • a compound of formula I as defined in the first aspect and any technically sensible combination of its embodiments, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of a fibrotic condition.
  • the fibrotic condition may be keloidal scarring and/or hypertrophic scarring.
  • a cell based method for the identification of compounds suitable to treat a fibrotic condition comprising:
  • the fibrotic condition may be keloidal scarring and/or hypertrophic scarring.
  • a method of treating and/or preventing fibrotic scarring in a subject comprising the steps of providing a therapeutically effective amount of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof to a subject in need thereof.
  • a use of RepSox and/or thiazovivin, or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for use in treating fibrotic scarring is provided.
  • the fibrotic condition may be keloidal scarring and/or hypertrophic scarring.
  • FIG. 1 (a) Design and mechanism of FNPs for imaging of FAPa. (b) Synthesis of FNP1 and FNP2. Reagents and conditions: (i) resorcinol, K2CO 3 , acetonitrile (ACN), 50 °C, 6 h; (ii) triphosgene, anhydrous dichloromethane (DCM), 25 °C, 0.5 h; (iii) A/,/ ⁇ /-disuccinimidyl carbonate, A/,/ ⁇ /-Diisopropylethylamine (DIPEA), anhydrous ACN, 25 °C, overnight (iv) N,N’- dimethylethylenediamine, DIPEA, anhydrous tetrahydrofuran (THF), 25 °C, 8 h; (v) CyOOCI, K2CO 3 , anhydrous DCM, 25 °C, 5 h.
  • ACN acetonitrile
  • DIPEA ace
  • Figure 6 Human skin tissue histology
  • (a) Cross-sectional view of skin sections implanted with KF and NDF cells, their FNP1 signal (purple signal, 710 nm) imaged with epifluorescence microscope. ⁇ r ⁇ ’ - epidermis layer,‘Der’ - dermis layer. Scale bar: 100 pm.
  • FIG. 7 FNP1 probe in cell culture
  • FIG. 8 Cross-section view of FNP1 distribution in epidermis-removed (left) and epidermis- intact skin (right). Skin sections in Phase contrast, 461 nm (blue), 570 nm (red, green), 710 nm (purple, from left - right) fluorescence emission channels. Scale bar: 100 pm.
  • FIG. 9 (a) Schematic illustration of microneedle-assisted penetration of FNP1 for FAPa imaging in keloid disease models (i) Skin tissue pre-treated with microneedles 10 to generate micro-channels 20 (5 min, 16.7 kPa pressure), (ii) micro-channels 20 facilitate FNP1 30 penetration, (iii) fluorescence imaging (b) Representative fluorescence imaging of unmodified skin, skin implanted with HaCaT, NDF or KF cells after treatment with FNP1 (20 mI_, 250 mM) for 6 h. (c) Quantification of fluorescence intensities of the skins from Figure 9b. The fluorescence intensities derived from FNP1 were normalized by total cell number (@ 570 nm).
  • FIG. 10 FNP1 performance validation in skin fibroblast cells
  • H&E staining of the abnormal scar tissue vertical lines denote margins of wound/scar (S), U: unwounded skin, E: epidermis (uppermost skin layer), D: dermis (lower skin layer);
  • FNP1 signals in NDF, KF, HSF and HaCaT with Hoechst 33342 signal as the reference signal (b) FAP-a gene expression in NDF, KF, HSF and HaCaT with GAPDH as the reference gene;
  • FNP1 probe for accessing TQRb modulation in fibroblast cells (a) FNP1 and Hoechst staining of untreated NDF, NDF treated with 2 ng/ml TQRb1 , untreated HSF, HSF treated with 25 mM RepSox, untreated KF, and KF treated with 25mM Repsox; (b) Fluorescence quantification in (a); (c) Fluorescence quantification of FNP1 signal in NDF treated with a range of TQRb1 (0.016, 0.08, 0.4, 2, 10, 50 ng/ml); (d) Correlation of FAP-a gene expression with normalized FNP1 signal. *: P ⁇ 0.05, **: P ⁇ 0.01.
  • FIG. 12 PCR quantification of FAPa mRNA expression in NDF following the TQRb1 treatment (0, 0.4, 2, 10, 50 ng/ml). FAPa mRNA expression was normalized by GAPDH mRNA expression.
  • FIG. 13 Drug screening with FNP1 probe: Normalized FNP1 signal and cell viability under different treatment conditions for (a) HSF and (b) KF. The different drugs are grouped to negative controls that have no anti-scarring response (negative), positive controls with known anti-scarring properties (positive), and drugs with unknown effects (drug screening). FNP1 (bar graph) and viability (line graph) signals read from the left and right axis respectively.
  • the treatment groups consist of UT (untreated), TGF (T ⁇ Rb1 , 2ng/ml), Rsox (RepSox, 25 mM), Rapa (Rapamycin, 20 nM), Dec (Decorin, 100 nM), Simv (Simvastatin, 20 mM), Perf (Pirfenidone, 50 pg/ml), Thia (Thiazovivin, 500 nM), DMSO (dimethyl sulfoxide, 2.5%), VPA (valproic acid, 500 mM), Tran (Tranylcypromine, 5 mM), PD (PD0325901 , 1 mM), FSK (forskolin, 10 mM), CHIR (CHIR99021 , 10 mM), Vitc (Vitamin C, 10 pg/ml).
  • Figure 15 Effects of newly-identified anti-scarring drugs on the cellular expression of (a) COL1A1 and (b) CTGF in KF and HSF after the treatment of Rsox, Thia, and CHIR. *:p ⁇ 0.05, **:p ⁇ 0.01.
  • Figure 16 PCR analysis of (a) COL1A1 and (b) CTGF expression in KF and HSF treated with drug candidates normalized to values from NDF (normal dermal fibroblasts).
  • FIG. 1 Effects of newly-identified anti-scarring drugs on the expression of a-SMA protein in UT (untreated), Thia (thiazovivin)-treated, Rsox (RepSox)-treated, Simv (simvastatin)- treated HSF and KF cells. Scale bars are: 20 pm.
  • FIG. 1 Immunostaining of a-smooth muscle actin (a-SMA) in untreated and TGF-bI treated normal dermal fibroblasts (NDFs). Scale bars are: 20 pm.
  • a-SMA a-smooth muscle actin
  • NIR near-infrared
  • FAPa fibroblast activation protein-alpha
  • ECM extracellular matrix
  • Said marker may play a role in other fibrotic conditions and so may also be useful in detecting such conditions too.
  • X represents a fluorophore group covalently linked to the rest of the molecule by way of an oxygen atom or an NH group;
  • Y is a self-immolative linking group
  • Z represents a peptide group of formula la:
  • the wavy line represents the point of attachment to the rest of the molecule
  • R 1 represents Ci-e alkyl or OR 2 ;
  • R 2 represents Ci-e alkyl that is unsubstituted or substituted by one or more groups selected from halo, C 1-3 alkyl and aryl;
  • n 1 or 2;
  • n 0 or 1 , or pharmaceutically acceptable salts or solvates thereof, provided that:
  • the compounds disclosed herein are selective (discriminating keratinocytes and normal fibroblasts from keloid derived fibroblasts) and sensitive (e.g. compounds disclosed herein may only need as few as 20,000 cells to provide a readout). Thus, the compounds disclosed herein show promise as both a diagnostic tool and as a screening tool.
  • the word“comprising” may be interpreted as requiring the features mentioned, but not limiting the presence of other features.
  • the word “comprising” may also relate to the situation where only the components/features listed are intended to be present (e.g. the word“comprising” may be replaced by the phrases“consists of” or“consists essentially of”). It is explicitly contemplated that both the broader and narrower interpretations can be applied to all aspects and embodiments of the present invention.
  • the word“comprising” and synonyms thereof may be replaced by the phrase“consisting of” or the phrase“consists essentially of’ or synonyms thereof and vice versa.
  • References herein (in any aspect or embodiment of the invention) to compounds of formula I includes references to such compounds per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such compounds.
  • salts include acid addition salts and base addition salts.
  • Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of a compound of formula I with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counter-ion of a compound of formula I in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.
  • Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2- sulphonic, naphthalene-1 , 5-disulphonic and p-toluenesulphonic), ascorbic (e.g.
  • L-glutamic L-glutamic
  • a-oxoglutaric glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic
  • lactic e.g. (+)-L-lactic and ( ⁇ )-DL- lactic
  • lactobionic maleic, malic (e.g.
  • salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or preferably, potassium and calcium.
  • mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids
  • organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids
  • metals such as sodium, magnesium, or preferably, potassium and calcium.
  • solvates are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the compounds of the invention of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent).
  • solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide.
  • Solvates can be prepared by recrystallising the compounds of the invention with a solvent or mixture of solvents containing the solvating solvent.
  • Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the compound to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.
  • TGE thermogravimetric analysis
  • DSC differential scanning calorimetry
  • X-ray crystallography X-ray crystallography
  • the solvates can be stoichiometric or non-stoichiometric solvates. Particularly preferred solvates are hydrates, and examples of hydrates include hemihydrates, monohydrates and di hydrates.
  • Compounds of formula I may contain double bonds and may thus exist as E ( Chrysler ) and Z (zusammen) geometric isomers about each individual double bond. All such isomers and mixtures thereof are included within the scope of the invention.
  • Compounds of formula I may contain one or more asymmetric carbon atoms and may therefore exhibit optical and/or diastereoisomerism.
  • Diastereoisomers may be separated using conventional techniques, e.g. chromatography or fractional crystallisation. The various stereoisomers may be isolated by separation of a racemic or other mixture of the compounds using conventional, e.g. fractional crystallisation or HPLC, techniques.
  • the desired optical isomers may be made by reaction of the appropriate optically active starting materials under conditions which will not cause racemisation or epimerisation (i.e. a‘chiral pool’ method), by reaction of the appropriate starting material with a‘chiral auxiliary’ which can subsequently be removed at a suitable stage, by derivatisation (i.e.
  • a resolution for example with a homochiral acid followed by separation of the diastereomeric derivatives by conventional means such as chromatography, or by reaction with an appropriate chiral reagent or chiral catalyst all under conditions known to the skilled person. All stereoisomers and mixtures thereof are included within the scope of the invention.
  • halo when used herein, includes references to fluoro, chloro, bromo and iodo.
  • aryl when used herein includes Ce-u (such as Ce-io) aryl groups. Such groups may be monocyclic, bicyclic or tricyclic and have between 6 and 14 ring carbon atoms, in which at least one ring is aromatic. The point of attachment of aryl groups may be via any atom of the ring system. However, when aryl groups are bicyclic or tricyclic, they are linked to the rest of the molecule via an aromatic ring. Ce-14 aryl groups include phenyl, naphthyl and the like, such as 1 ,2,3,4-tetrahydronaphthyl, indanyl, indenyl and fluorenyl. Embodiments of the invention that may be mentioned include those in which aryl is phenyl.
  • alkyl refers to an unbranched or branched, cyclic, saturated or unsaturated (so forming, for example, an alkenyl or alkynyl) hydrocarbyl radical, which may be substituted or unsubstituted (with, for example, one or more halo atoms).
  • alkyl refers to an acyclic group, it is preferably Ci-e alkyl (such as ethyl, propyl, (e.g. n-propyl or isopropyl), butyl (e.g. branched or unbranched butyl), pentyl or, more preferably, methyl).
  • alkyl is a cyclic group (which may be where the group “cycloalkyl” is specified), it is preferably C3-12 cycloalkyl and, more preferably, C5-10 (e.g. C5-7) cycloalkyl.
  • the term“alkyl” may be used to refer to unbranched or branched, saturated hydrocarbyl radicals that are substituted or, more particularly, unsubstituted.
  • fluorophore is intended to refer to a substituent group that does not fluoresce when attached to the rest of the molecule by way of a covalent bond, but which is capable of fluorescence following cleavage of said covalent bond.
  • the compounds of formula I contain a fluorophore that is initially nonfluorescent because the fluorophore is in a “caged” state due to the covalent bonds reducing the donation of electrons to the fluorophore system.
  • fluorophore X may in particular embodiments be independently selected from the group consisting of:
  • X may be independently selected from the group consisting of:
  • the peptide group Z may have the generic structure of formula la. As will be appreciated, the presence of a peptide group of this generic structure acts as a substrate for FAPa and so is required to be present to induce fluorescence of the fluorophore if there are elevated levels of the FAPa enzyme, which indicates the presence of a fibrotic condition.
  • Z may be independently selected from the group consisting of:
  • self-immolative linker is a bifunctional or a trifunctional chemical moiety which is capable of covalently linking together two or three spaced chemical moieties into a normally stable tri- or tetrapartite molecule, that can release one of the spaced chemical moieties from the stable molecule by means of enzymatic cleavage and following enzymatic cleavage, can spontaneously cleave from the remainder of the molecule to release the other spaced chemical moiety(ies).
  • An example of this cleavage process is provided in Figure 1a.
  • Any suitable self-immolative linker may be used in the current invention. Examples of suitable self-immolative linkers include, but are not limited to those selected from:
  • the group Y may be selected from the group of:
  • n there will be two Z groups. This may help to amplify the fluorescent signal produced (in the case where the same amounts of a compound where m is 2 is used compared to where n is 1), or may allow for less of the compound of formula I to be provided to a subject.
  • Examples of the compound of formula I include, but are not limited to those listed below.
  • the compound formula I (and salts and solvates thereof) may be selected from:
  • the compound of formula I may be selected from:
  • Compounds of formula I may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form.
  • Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
  • Compounds of formula I will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable adjuvant, diluent or carrier may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy , 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
  • the amount of compound of formula I in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the particular patient to be diagnosed, as well as the compound(s) which is/are employed. In any event, the amount of compound of formula I in the formulation may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • a controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.
  • a parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • a liquid or semisolid carrier or vehicle e.g. a solvent such as water
  • one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • compounds of formula I may be administered at varying diagnostically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a diagnostic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature the condition being diagnosed, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be diagnosed.
  • Administration may be continuous or intermittent (e.g. by bolus injection).
  • the dosage may also be determined by the timing and frequency of administration.
  • the dosage can vary from about 0.01 mg to about 1000 mg per day of a compound of formula I.
  • the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
  • the above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • compounds of formula I may be cleaved in the presence of FAPa and may therefore have utility as diagnostic agents for determining the presence and/or location (either in vivo or in vitro) of a fibrotic condition.
  • a method of detecting a fibrotic condition in a subject comprising the steps of providing a compound of formula I as defined above, or a pharmaceutically acceptable salt or solvate thereof to a subject, subjecting a tissue or organ suspected of suffering from a fibrotic condition to irradiation with light and detecting fluorescence from the irradiated tissue or organ, wherein an increase in fluorescence compared to a control indicates the presence of a fibrotic condition.
  • a compound of formula I, as defined above, or a pharmaceutically acceptable salt or solvate thereof for use in the detection of a fibrotic condition.
  • fibrotic condition is intended to cover dermal fibrosis (e.g. hypertrophic scars, keloids, bums, Peyronie's disease, and Dupuytren's contractures) and non-dermal fibrosis (e.g. lung (or pulmonary) fibrosis, liver/hepatic fibrosis, ocular fibrosis, fibrosis of the gut, kidney/renal fibrosis, pancreatic fibrosis, vascular fibrosis, cardiac fibrosis, and myelofibrosis).
  • dermal fibrosis e.g. hypertrophic scars, keloids, bums, Peyronie's disease, and Dupuytren's contractures
  • non-dermal fibrosis e.g. lung (or pulmonary) fibrosis, liver/hepatic fibrosis, ocular fibrosis, fibrosis of the gut, kidney/renal fibrosis, pancreatic fibros
  • Fibrotic conditions of the lung include, but are not limited to, idiopathic pulmonary fibrosis (“IFF”); idiopathic pulmonary upper lobe fibrosis (Amitani disease); familial pulmonary fibrosis; pulmonary fibrosis secondary to systemic inflammatory diseases such as, rheumatoid arthritis, scleroderma, lupus, cryptogenic fibrosing alveolitis, chronic obstructive pulmonary disease (“COPD”) or chronic asthma or secondary to radiation exposure; cystic fibrosis; non-specific interstitial pneumonia (“NSIP”); cryptogenic organizing pneumonia (“COP”); progressive massive fibrosis, a complication of coal worker's pneumoconiosis; scleroderma/systemic sclerosis; bronchiolitis obliterans-organizing pneumonia; pulmonary hypertension; pulmonary tuberculosis; silicosis; asbestosis; acute lung injury; and acute
  • Fibrotic conditions of the liver include, but are not limited to, liver cirrhosis due to all etiologies; congenital hepatic fibrosis; obesity; fatty liver; alcohol induced liver fibrosis; non-alcoholic steatohepatitis (NASH); biliary duct injury; primary biliary cirrhosis; infection- or viral-induced liver fibrosis (e.g., chronic hepatitis B and C virus infections); cystic fibrosis; autoimmune hepatitis; necrotizing hepatitis; primary sclerosing cholangitis; hemochromatosis; disorders of the biliary tree; hepatic dysfunction attributable to infections; and fibrosis secondary to radiation exposure.
  • liver fibrosis due to all etiologies
  • congenital hepatic fibrosis e.e., obesity
  • fatty liver fatty liver
  • alcohol induced liver fibrosis non-alcoholic steato
  • Fibrotic conditions of the heart and/or pericardium include, but are not limited to, endomyocardial fibrosis; cardiac allograft vasculopathy (“CAV”); myocardial infarction; atrial fibrosis; congestive heart failure; arterioclerosis; atherosclerosis; vascular stenosis; myocarditis; congestive cardiomyopathy; coronary infarcts; varicose veins; coronary artery stenosis and other post- ischemic conditions; and idiopathic retroperitoneal fibrosis.
  • CAV cardiac allograft vasculopathy
  • Fibrotic conditions of the kidney include, but are not limited to, glomerulonephritis (including membranoproliferative, diffuse proliferative, rapidly progressive or sclerosing, post-infectious and chronic forms); diabetic glomerulosclerosis; focal segmental glomerulosclerosis; IgA nephropathy; diabetic nephropathy; HIV-associated nephropathy; membrane nephropathy; glomerulonephritis secondary to systemic inflammatory diseases such as lupus, scleroderma and diabetes glomerulonephritis; idiopathic membranoproliferative glomerular nephritis; mesangial proliferative glomerulonephritis; crescentic glomerulonephritis; amyloidosis (which affects the kidney among other tissues); autoimmune nephritis; renal tubuloinsterstitial fibrosis; renal
  • Fibrotic conditions of the pancreas include, but are not limited to, stromal remodeling pancreatitis and stromal fibrosis.
  • Fibrotic conditions of the gastrointestinal tract include, but are not limited to, Crohn's disease; ulcerative colitis; collagenous colitis; colorectal fibrosis; villous atrophy; crypt hyperplasia; polyp formation; healing gastric ulcer; and microscopic colitis.
  • Fibrotic conditions of the eye include, but are not limited to, ocular fibrosis, ophthalmic fibrosis, proliferative vitreoretinopathy; vitreoretinopathy of any etiology; fibrosis associated with retinal dysfunction; fibrosis associated with wet or dry macular degeneration; scarring in the cornea and conjunctiva; fibrosis in the corneal endothelium; anterior subcapsular cataract and posterior capsule opacification; anterior segment fibrotic diseases of the eye; fibrosis of the corneal stroma (e.g., associated with corneal opacification); fibrosis of the trabecular network (e.g., associated with glaucoma); posterior segment fibrotic diseases of the eye; fibrovascular scarring (e.g., in retinal or choroidal vasculature of the eye); retinal fibrosis; epiretinal fibrosis; retinal gliosis; subretinal fibrosis (e.
  • Additional fibrotic disorders or fibrosis resulting from any one of the aforementioned conditions include, but are not limited to, spinal cord injury/fibrosis or central nervous system fibrosis such as fibrosis after a stroke, fibrosis associated with neurodegenerative disorder such as Alzheimer's disease or multiple sclerosis; vascular restenosis; uterine fibrosis; endometriosis; ovarian fibroids; Peyronie's disease; polycystic ovarian syndrome; disease related pulmonary apical fibrosis in ankylosing spondylitis; and fibrosis incident to microbial infections (e.g., bacterial, viral, parasitic, fungal etc.).
  • microbial infections e.g., bacterial, viral, parasitic, fungal etc.
  • the fibrotic condition for diagnosis may be keloidal scarring and/or hypertrophic scarring.
  • aspects of the invention described herein may have the advantage that, in the diagnosis of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, be more selective than, be more sensitive than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the diagnosis of those conditions or otherwise.
  • a cell based method for the identification of compounds suitable to treat a fibrotic condition comprising:
  • Examples of cells expressing a fibrotic condition include activated fibroblasts such as keloid- derived fibroblasts (KF) and fibroblasts derived from hypertrophic scar (HSFs) which, compared to normal fibroblasts, overexpress fibroblast activation protein-alpha (FAPa) and other biomarkers, which may include, but are not limited to, Col1a1 , CTGF, fibronectin, a- smooth muscle actin(SMA), TQRb-Gbobr ⁇ qG I etc. Cells which are genetically engineered to overexpress FAPa may also be considered to express a fibrotic condition.
  • activated fibroblasts such as keloid- derived fibroblasts (KF) and fibroblasts derived from hypertrophic scar (HSFs) which, compared to normal fibroblasts, overexpress fibroblast activation protein-alpha (FAPa) and other biomarkers, which may include, but are not limited to, Col1a1 , CT
  • the screening method disclosed herein has been used to identify two compounds that show an anti-fibrotic effect, namely RepSox and thiazovivin. It is noted that the screening method disclosed herein is phenotypic in nature, as RepSox and thiazovivin have different mechanisms of action. Thus the screening test is able to work across a range of different potential mechanisms of action and may be useful in a drug discovery programme for new chemical entities that act as anti-fibrotic agents, or in screening existing drugs that are known to be safe and well-tolerated for anti-fibrotic activity.
  • RepSox and/or thiazovivin can be applied just after wound closure and prior to the emergence of an abnormal scar lesion in a subject who is susceptible to such scarring.
  • the subject in question also has existing abnormal scarring as the result of a fibrotic condition, then these scars will be treated at the same time as new fibrotic scarring is prevented.
  • the test methods described herein may be used to establish if the subject is susceptible to such scarring, which can then be prevented accordingly.
  • the use of RepSox and/or thiazovivin can be used to treat these existing scars.
  • any of the fibrotic conditions mentioned herein may benefit from these compounds to treat said conditions.
  • particular conditions that may benefit include, but are not limited to, keloidal scarring and/or hypertrophic scarring.
  • RepSox and/or thiazovivin may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form.
  • Particular modes of administration that may be mentioned include oral, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration.
  • RepSox and/or thiazovivin will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice.
  • a pharmaceutically acceptable adjuvant diluent or carrier
  • Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use.
  • Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995).
  • a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.
  • any pharmaceutical formulation(s) used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount of RepSox and/or thiazovivin (and suitable salts and solvates) in the formulation(s) may be determined routinely by the skilled person.
  • a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment.
  • a controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer.
  • a parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • a liquid or semisolid carrier or vehicle e.g. a solvent such as water
  • one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.
  • RepSox and/or thiazovivin may be administered at varying therapeutically effective doses to a patient in need thereof.
  • the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe.
  • the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.
  • Administration may be continuous or intermittent (e.g. by bolus injection).
  • the dosage may also be determined by the timing and frequency of administration.
  • the dosage can vary from about 0.01 mg to about 1000 mg per day of RepSox and/or thiazovivin (and suitable salts and solvates).
  • the medical practitioner or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient.
  • the above- mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.
  • RepSox and/or thiazovivin act by differing mechanisms of action. Therefore, the compounds may be used individually as monotherapies, or may be combined together to provide a combination therapy. In this circumstance, the combined therapy may be provided as a single formulation (e.g. a single pill) or as separate formulations that may be administered sequentially, simultaneously or concomitantly, as determined by a skilled physician.
  • the aspects of the invention described herein in relation to RepSox and thiazovivin may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.
  • normal dermal fibroblasts NDFs
  • fibroblasts derived from human keloid scar tissue KFs
  • fibroblasts derived from human hypertrophic scar tissue HSFs
  • immortalized keratinocyte HaCaT
  • cells were stained with FNP1 at 2 mM final concentration in culture medium for 30 min. Excess probes were rinsed off twice with PBS, prior to nuclear staining with Hoescht 33342 for 15min as in manufacturer’s protocol. Fluorescence images of cells were taken with Laser Scanning Microscope LSM800 (Zeiss) or LX71-inverted fluorescence microscope (Olympus) with Retiga-2000R CCD camera. Throughout the experiments, image capture settings were kept constant for FNP channel (500 ms, 8x gain) and Hoechst 33342 channel (50 ms, 3x gain) with 100x magnification for respective fluorescence channels and transmitted light channel. Background fluorescence was removed with ImageJ software.
  • T-test or one-way ANOVA was carried out to calculate the significance P-value.
  • a suitable post-hoc test was chosen using the IBM SPSS Statistics 22 software. For each experiment, values are reported as mean ⁇ standard deviation of at least 3 independent samples.
  • A/,/ ⁇ / -Disuccinimidyl carbonate (DSC, 96 mg, 0.375 mmol) was dissolved in anhydrous CH3CN. Then, the mixture of compound 1-1 (60 mg, 0.15 mmol) and DIPEA (76.5 mI_, 0.45 mmol) were added via a syringe. The resulting reaction mixture was stirred overnight at room temperature under N2 atmosphere. After reaction, the solvent was removed under reduced pressure and the product was used in the next step without further purification. Such crude active carbonate mixture was added to the solution of N,N’- dimethylethylenediamine (132 mg, 1.5 mmol) and DIPEA (132 mg, 1.5 mmol) in anhydrous THF.
  • Hemicyanine (CyOH, 40 mg, 0.10 mmol; form preparation 1) was added dropwise to the solution of triphosgene (15 mg, 0.05 mmol) in anhydrous CH2CI2 at 0 °C under N2 atmosphere. After reaction at room temperature for 0.5 h, comminuted K 2 CC> 3 (138 mg, 1.0 mmol) was added and stirred for another 0.5 h. Then, compound 3-1 (42 mg, 0.08 mmol) was added dropwise and the reaction mixture was stirred for 5 h at room temperature under N2 atmosphere and the pure product FNP1 (46 mg, 62%) was obtained after HPLC purification.
  • A/,/ ⁇ /-Disuccinimidyl carbonate (DSC, 96 mg, 0.375 mmol) was dissolved in anhydrous CH 3 CN. Then, the mixture of compound 1-2 (48 mg, 0.15 mmol) and DIPEA (76.5 mI_, 0.45 mmol) were added via a syringe. The resulting reaction mixture was stirred overnight at room temperature under N 2 atmosphere . After reaction, the solvent was removed under reduced pressure and the product was used in the next step without further purification. Such crude active carbonate mixture was added to the solution of N,N’- dimethylethylenediamine (132 mg, 1.5 mmol) and DIPEA (132 mg, 1.5 mmol) in anhydrous THF.
  • Hemicyanine (CyOH, 40 mg, 0.10 mmol; from Preparation 1) was added dropwise to the solution of triphosgene (15 mg, 0.05 mmol) in anhydrous CH2CI2 at 0 °C under N 2 atmosphere. After reaction at room temperature for 0.5 h, comminuted K 2 CC> 3 (138 mg, 1.0 mmol) was added and stirred for another 0.5 h. Then, compound 3-2 (35 mg, 0.08 mmol) was added dropwise and the reaction mixture was stirred for 5 h at room temperature under N2 atmosphere and the pure product FNP2 (46 mg, 62%) was obtained after HPLC purification.
  • FNP2 showed subtle change in its absorption even after incubation with FAPa for 120 min ( Figure 2a and Figure 3c).
  • the ratiometric absorption signal A682/A590 (the ratio of the absorption intensity at 682 nm to that at 590 nm) was quantified as a function of incubation time at different concentrations of FAPa ( Figure 3b).
  • FNP1 showed increased A682/A590 with increased incubation time and reached its plateau at 25 min, indicating the complete conversion of FNP1 into free CyOH. At this time point, FNP1 showed 45-fold enhancement in the fluorescence intensity at 710 nm, which was only 10-fold for FNP2. ( Figure 2b).
  • V ⁇ nax*[S] (Km + [S]) where V is initial velocity, and [S] is substrate concentration.
  • FNP1 catalytic efficiencies (, k cat /K m ) of FAPa towards FNP1 was calculated to be 1.64 x 10 4 ⁇ 1.04 x 10 3 M 1 s -1 , 38.1-fold higher than that of FNP2.
  • FNP1 activation was tested against FAPa in the presence of its inhibitor Val-boroPro (talabostat) or other enzymes relevant to skin diseases including dipeptidyl peptidase IV (DPPIV), matrix metalloproteinase (MMP)-1 , MMP-2, MMP- 13, caspase-3, and tissue plasminogen activator (tPA).
  • DPPIV dipeptidyl peptidase IV
  • MMP matrix metalloproteinase
  • tPA tissue plasminogen activator
  • NDF Normal dermal fibroblasts
  • KF keloid-derived fibroblasts
  • HaCaT immortalized keratinocyte
  • the cells were washed three times using PBS buffer (10 mM, pH 7.4) and then incubated with FAP-1 (5 mM) for 1 h at 37 °C in an atmosphere of 5% CO2 and 95% humidified air. After incubation, the medium was removed and the cells were washed 3 times using PBS buffer (10 mM, pH 7.4). Then the cells were stained with lysotracker (LysoTracker®, Thermo Fisher) for the lysosome and with Hoechst 33342 (NucBlue Live ReadyProbes Reagent, Thermo Fisher) for the nuclei as protocol.
  • Excitation/emission wavelengths were 350 ⁇ 20/460 ⁇ 20 nm for Hoechst 33342, 488 ⁇ 20/525 ⁇ 20 nm for lysosome indicator, and 680 ⁇ 20/710 ⁇ 20 nm for FNP1. Fluorescence imaging
  • Fluorescence images of cells were acquired using LX71-inverted fluorescence microscope (Olympus) with Retiga-2000R CCD camera ( Figure 7b). Throughout all the experiments, capture settings were fixed for FNP1 channel (400ms, 8x gain), Dil channel (100 ms, 3 x gain) and Hoescht33342 channel (40 ms, 3 x gain). ImageJ software was utilized to remove signal background and quantify cellular fluorescence intensity. Confocal imaging was performed on Laser Scanning Microscope LSM800 (Zeiss), with 200 x magnification for respective fluorescence channels and transmitted light channel ESID ( Figure 7a). Z-stack images of 2 pm were taken on fixed cells after treatment with 10% neutral-buffered formalin (NBF) for 10 min.
  • NBF neutral-buffered formalin
  • NDF 4 x 10 5 NDF, KF, HaCaT or TGF-bI -treated NDF were resuspended and lysed with TRIzol reagent. Following RNA extraction, cDNA conversion was performed with M-MLV Rnase H(-) Mutant kit. Primer sequences for FAP-a and GAPDH mRNA are listed in Table 3. CT values of both genes were obtained through quantitative PCR steps on CFX ConnectTM PCR System (Biorad). 2-AACT formula was utilized to compare FAP-a expression level between groups (normalized against NDF).
  • FNP1 was then applied to detect KF cells in vitro along with several control skin cells including: HaCaT (epidermis origin) and normal dermal fibroblasts (NDF, dermis origin). After a short incubation period (1 h), strong NIR fluorescence was detected for KF (Figure 7a). Co-staining studies confirm that FNP1 signal was mainly localized in the cytoplasm, including the cell lysosome. In contrast, weak fluorescence signal was observed in other cells including HaCaT and NDF ( Figure 7b).
  • NIR fluorescence of FNP1 in KF cells was 19.2 and 2.23-fold higher than in HaCaT and NDF, respectively ( Figure 7c).
  • NDF cells were stimulated using transforming growth factor (TGF) ⁇ 1 , which is known to increase FAPa expression levels.
  • TGF transforming growth factor
  • the NIR fluorescence of FNP1 in NDF cells was enhanced by 4.15-fold after TGF-bI stimulation, confirming that the higher NIR signal in KF cells was a result of its higher FAPa expression levels relative to normal skin cells (i.e. NDF).
  • FNP1 The ability of FNP1 to detect KF cells was subsequently evaluated in live, metabolically- active human skin tissue models containing diseased KF cells as a proof-of-concept.
  • FNP1 was mixed with Aquaphor® ointment to form an emulsion to help it cross the uppermost skin epidermal barrier to interact with dermis residing KF cells for topical application.
  • microneedles 10 were employed to create microchannels 20 ( Figure 9ai).
  • Microneedle device 500 pm in height per needle
  • the KF cells 60 were located at the dermis layer 50.
  • the microneedles were weighted down to deform skin at 18-fold pressure magnitude below that required to break skin (i.e. 300 kPa).
  • the FNP1 probe is a highly specific, easy-to-use diagnostic strategy suitable to provide early indications of abnormal scarring before progression into mature keloid scars.
  • FNPTs sensitivity was further evaluated by applying it to normal dermal fibroblasts (NDF), keloid fibroblasts (KF), hypertrophic scar fibroblasts (HSF) and keratinocytes (HaCaT, non expressing cells), by the procedures set out above in the materials and methods section. This was compared to conventional gene expression analysis, PCR.
  • NDF normal dermal fibroblasts
  • KF keloid fibroblasts
  • HSF hypertrophic scar fibroblasts
  • HaCaT keratinocytes
  • Fibroblasts derived from abnormal (keloid, hypertrophic) scar lesions (Figure 10a,‘S’ region) express higher FAPa than normal dermal fibroblasts (NDFs) derived from undiseased skin.
  • NDFs normal dermal fibroblasts
  • HSF, KF hypertrophic, keloid scar-derived fibroblasts
  • keratinocytes cells derived from the epidermis layer, HaCaT, Figure 10a, ⁇ ’ region
  • the FNP1 signal was obtained at excitation: 680 nm, emission: 710 nm using a fluorescence multi-plate reader. NDF, KF, HSF and HaCaT were evaluated. Being of skin epidermal lineage, HaCaT cells, devoid of FAPa, express a weak FNP1 signal (Figure 10b). NDF produced a fluorescence signal 2.4-fold higher by comparison. However, the FNP1 signal was 5.76-fold and 4.32-fold higher in keloids and hypertrophic scar fibroblasts respectively (compared to HaCaT). When considering NDF as the baseline, FNP1 signals from KF and HSF were >1.7-fold and >1.4-fold higher respectively than that from NDF (Figure 10b).
  • FNP1 signal in HaCaT was 0.41 folds of that in NDF.
  • PCR analysis of FAPa expression trended in a similar way to FNP1 analysis.
  • FAPa in KF, HSF, HaCaT was 7.3-fold, 8-fold and 0.6-folds of that in NDF respectively ( Figure 10c).
  • the indirect quantification of FAPa using FNP1 method was comparable to the direct quantification of FAPa expression with PCR (R 2 , coefficient of determination was 0.8712, Figure 10d).
  • the FNP1 probe can discriminate between skin cells with differential FAPa expression and this confirms the reliability of FNP1 assay to quantify cellular FAPa.
  • NDFs (with low endogenous FAPa expression and TQRb activity) were titrated with TQRb1 using a range of concentrations (0.016 - 50 ng/ml, Figure 11 c).
  • Normalized FNP1 probe signal was significantly higher in 10 and 50 ng/ml TQRb1 compared to untreated NDF. This was found to be 3.07 and 2.89-fold higher respectively.
  • the smallest quantity of TQRb1 (0.016 ng/ml) resulted in a 1.57-fold increase in FNP1 signal demonstrating FNP1 sensor sensitivity (Figure 11 c).
  • an ascending trend in FNP1 signal with respect to increasing TQRb1 concentration was observed.
  • Even 0.016 ng/ml - 3,125-fold lower than the maximum TQRb1 concentration showed an increase in FNP1 signal.
  • FNP1 The quantification of FAPa using the FNP1 method was comparable to the PCR quantification (R 2 was 0.8527, Figure 11 d).
  • R 2 was 0.8527, Figure 11 d.
  • FNP1 is highly sensitive, affirming it can detect changes in FAPa induced by changes in TQRb activity.
  • the FNP1 probe may further identify anti-scarring drugs caused by mechanisms besides TQRb.
  • FNP x and FNPo represent the FNP intensities of the cells from treatment‘X’ and for the untreated cells, respectively.
  • NDFs, KFs or HSFs were rinsed with PBS and fixed with 4% paraformaldehyde in PBS for 10 minutes on ice. Fixed cells were then stained with monoclonal Anti-Actin, a-Smooth Muscle- Cy3 clone 1A4 overnight at 4 °C, with final concentration of 6 pg/ml. Following thorough rinsing with chilled PBS, Hoescht 33342 staining was performed before confocal microscopy imaging. Immunostaining and imaging were performed in triplicate and repeated.
  • the identified Rsox and Thia suppressed FAP expression were further evaluated using the following assays: wound healing model (scratch assay), gene expression analysis and immunofluorescence imaging of known abnormal scar biomarkers.
  • COL1A1 (Figure 15a) is responsible for excess scar tissue generation and CTGF (Figure 15b) is an activation target of T ⁇ Rb signaling activity.
  • Keloid and hypertrophic scars express abnormally high COL1A1 and CTGF levels.
  • COL1A1 expressions in KSFs and HSFs were 5.8-fold and 5.1-fold higher than that in NDFs respectively ( Figure 16a).
  • Treatment with Rsox and Thia led to significant decreases in COL1A1 expression levels (85% and 87%) in HSF and KSF cells respectively ( Figure 15a).

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Abstract

L'invention concerne un composé de formule I : [X]n-[Y]m-Z, dans laquelle X représente un groupe fluorophore lié par covalence au reste de la molécule au moyen d'un atome d'oxygène d'un groupe NH ; Y est un groupe de liaison auto-immolateur ; Z représente un dipeptide Gly-Pro bloqué par N ; n est compris entre 1 et 2 ; et m est compris entre 0 et 1. Le fluorophore X n'est pas fluorescent lorsqu'il est fixé au reste de la molécule par liaison covalente, mais est capable de fluorescence après le clivage de ladite liaison covalente. Les composés de formule I sont utiles comme sonde fluorescente dans le diagnostic de troubles fibrotiques in vivo, tels qu'une cicatrice chéloïde et/ou hypertrophique, et peuvent également être utilisés dans un test in vitro pour cribler des composés utiles pour traiter de tels troubles fibrotiques. L'invention concerne également l'utilisation de RepSox et de thiazovivine, qu'ils soient seuls ou en association, pour traiter des troubles fibrotiques.
PCT/SG2018/050608 2017-12-12 2018-12-12 Sondes moléculaires en proche infrarouge destinées à être utilisées dans le diagnostic de troubles fibrotiques et le criblage de médicaments anti-fibrotiques WO2019117812A1 (fr)

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CN114591632A (zh) * 2022-01-23 2022-06-07 大连理工大学 一类氮杂吲哚-半花菁染料、其合成方法及应用

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